The incidence of antibiotic resistance is increasing in many groups of disease-causing bacteria. Although some bacteria become resistant to antibiotics through mutation, acquisition of antibiotic resistance genes from other bacteria is probably more common. This proposal focuses on the genus Bacteroides. Bacteroides species are among the numerically predominant species of bacteria in the human colon. Bacteroides species are also opportunistic pathogens that can cause life-threatening infections. Many Bacteroides strains have become resistant to multiple antibiotics. Transfer of resistance genes among these strains appears to have occurred mainly through the actions of a group of conjugative transposons (CTns), represented by CTnDOT. CTnDOT excises from the chromosome to form a circular intermediate, which transfers by conjugation to a recipient and integrates into the recipient's chromosome. During the previous funding period, genes responsible for excision and transfer were identified and shown to be controlled by a complex set of regulatory genes. These genes may allow the CTn to coordinate excision and transfer so that nicking to initiate transfer of the circular form does not occur until excision is complete. Both excision and transfer are stimulated over 1,000-fold by the antibiotic tetracycline. Previously, we found that three genes, rteA, rteB and rteC function as central regulatory genes. We propose that RteC triggers expression of two excision genes, excA and excB. The first specific aim of the proposal is to test this hypothesis. The second aim is to determine how ExcA and ExcB, presumably in concert with the CTn integrase (Int), catalyze excision and circularization of the CTn. The third specific aim is to determine whether RteC also controls expression of a gene currently designated as orf5 and to test the hypothesis that Orf5 in turn controls the expression of transfer (tra) genes. The fourth specific aim is to answer the question of how effective coordination of excision and transfer actually is. The last aim is to define the characteristics and functions of RteA and RteB, which control expression of rteC.